Recent developments in ion-trap mass spectrometry and related technologies

Due to their versatility, quadrupole ion traps have become popular mass spectrometers in the growing field of proteomics. High sensitivity, user friendliness and low cost are the key features that have contributed to the success of the technology. However, mass measurement accuracy, resolution and mass range are still not comparable to the analytical performances obtained on other mass spectrometers. In the past 5 years, researchers have tried to overcome these drawbacks, focusing their attention on two different aspects of ion-trap mass spectrometry, development of novel types of ion traps and manipulation of the gas-phase ion chemistry, in order to obtain alternative techniques for tandem mass spectrometry analysis. In the field of trapping devices, improvements in instrumental design have led to the linear ion trap, digital ion trap and orbitrap. Activation methods based on electrons, chemically produced by an anion or from irradiation with an electron beam, have demonstrated their utility in providing complementary sequence information for improving confidence in protein identification.     

Introduction to ion trap mass spectrometry: Application to the structural characterization of plant oligosaccharides

This review will focus on ion trap mass spectrometry (ITMS) and the application of this technique to the structural analysis of carbohydrates. The basic principles of operation of the electrostatic ion traps are briefly described and the applicability of the technique to the structural characterization of carbohydrates is illustrated with the analysis of arabinoxylan oligosaccharides by ion trap mass spectrometry.     

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.     

Differentiation of the anomeric configuration and ring form of glucosyl-glycolaldehyde anions in the gas phase by mass spectrometry: isomeric discrimination between m/z 221 anions derived from disaccharides and chemical synthesis of m/z 221 standards

Mass spectrometry of disaccharides in the negative-ion mode frequently generates product anions of m/z 221. With glucose-containing disaccharides, dissociation of isolated m/z 221 product ions in a Paul trap yielded mass spectra that easily differentiated between both anomeric configurations and ring forms of the ions. These ions were shown to be glucosyl-glycolaldehydes through chemical synthesis of their standards. By labeling the reducing carbonyl oxygen of disaccharides with 18O to mass discriminate between monosaccharides, it was established that the m/z 221 ions are comprised solely of an intact nonreducing sugar with a two-carbon aglycon derived from the reducing sugar, regardless of the disaccharide linkage position. This enabled the anomeric configuration and ring form of the ion to be assigned and the location of the ion to the nonreducing side of a glycosidic linkage to be ascertained. Detailed studies of experimental factors necessary for reproducibility in a Paul trap demonstrated that the unique dissociation patterns that discriminate between the isomeric m/z 221 ions could be obtained from month-to-month in conjunction with an internal energy-input calibrant ion that ensures reproducible energy deposition into isolated m/z 221 ions. In addition, MS/MS fragmentation patterns of disaccharide m/z 341 anions in a Paul trap enabled linkage positions to be assigned, as has been previously reported with other types of mass spectrometers.     

The standard protein mix database: a diverse data set to assist in the production of improved Peptide and protein identification software tools

Tandem mass spectrometry (MS/MS) is frequently used in the identification of peptides and proteins. Typical proteomic experiments rely on algorithms such as SEQUEST and MASCOT to compare thousands of tandem mass spectra against the theoretical fragment ion spectra of peptides in a database. The probabilities that these spectrum-to-sequence assignments are correct can be determined by statistical software such as PeptideProphet or through estimations based on reverse or decoy databases. However, many of the software applications that assign probabilities for MS/MS spectra to sequence matches were developed using training data sets from 3D ion-trap mass spectrometers. Given the variety of types of mass spectrometers that have become commercially available over the last 5 years, we sought to generate a data set of reference data covering multiple instrumentation platforms to facilitate both the refinement of existing computational approaches and the development of novel software tools. We analyzed the proteolytic peptides in a mixture of tryptic digests of 18 proteins, named the "ISB standard protein mix", using 8 different mass spectrometers. These include linear and 3D ion traps, two quadrupole time-of-flight platforms (qq-TOF), and two MALDI-TOF-TOF platforms. The resulting data set, which has been named the Standard Protein Mix Database, consists of over 1.1 million spectra in 150+ replicate runs on the mass spectrometers. The data were inspected for quality of separation and searched using SEQUEST. All data, including the native raw instrument and mzXML formats and the PeptideProphet validated peptide assignments, are available at http://regis-web.systemsbiology.net/PublicDatasets/.     

Ion mobility mass spectrometry for peptide analysis

The use of ion mobility mass spectrometry has grown rapidly over the last two decades. This powerful analytical platform now forms an attractive prospect for comprehensive analysis of many different molecular species, including chemically complex biological molecules. This paper describes the application of IM-MS to the study of peptides. We focus on three different ion mobility devices that are most frequently found in tandem with mass spectrometers. These are instruments using linear drift tubes (LDT), those using travelling wave ion guides (TWIGS) and those employing high field asymmetric ion mobility spectrometry (FAIMS). Each technique is described. Examples are given on the use of IM-MS for the determination of peptide structure, the study of peptides that form amyloid fibrils, and the study of complex peptide mixtures in proteomic investigations. We describe and comment on the methodologies used and the outlook for this developing analytical technique.     

Charge state estimation for tandem mass spectrometry proteomics

High-throughput protein analysis by tandem mass spectrometry produces anywhere from thousands to millions of spectra that are being used for peptide and protein identifications. Though each spectrum corresponds only to one charged peptide (ion) state, repetitive database searches of multiple charge states are typically conducted since the resolution of many common mass spectrometers is not sufficient to determine the charge state. The resulting database searches are both error-prone and time-consuming. We describe a straightforward, accurate approach on charge state estimation (CHASTE). CHASTE relies on fragment ion peak distributions, and by using reliable logistic regression models, combines different measurements to improve its accuracy. CHASTE's performance has been validated on data sets, comprised of known peptide dissociation spectra, obtained by replicate analyses of our earlier developed protein standard mixture using ion trap mass spectrometers at different laboratories. CHASTE was able to reduce number of needed database searches by at least 60% and the number of redundant searches by at least 90% virtually without any informational loss. This greatly alleviates one of the major bottlenecks in high throughput peptide and protein identifications. Thresholds and parameter estimates can be tailored to specific analysis situations, pipelines, and instrumentations. CHASTE was implemented in Java GUI-based and command-line-based interfaces.     

Recent developments and applications of electron transfer dissociation mass spectrometry in proteomics

Electron transfer dissociation (ETD) has been developed recently as an efficient ion fragmentation technique in mass spectrometry (MS), being presently considered a step forward in proteomics with real perspectives for improvement, upgrade and application. Available also on affordable ion trap mass spectrometers, ETD induces specific N–Cα bond cleavages of the peptide backbone with the preservation of the post-translational modifications and generation of product ions that are diagnostic for the modification site(s). In addition, in the last few years ETD contributed significantly to the development of top-down approaches which enable tandem MS of intact protein ions. The present review, covering the last 5 years highlights concisely the major achievements and the current applications of ETD fragmentation technique in proteomics. An ample part of the review is dedicated to ETD contribution in the elucidation of the most common posttranslational modifications, such as phosphorylation and glycosylation. Further, a brief section is devoted to top-down by ETD method applied to intact proteins. As the last few years have witnessed a major expansion of the microfluidics systems, a few considerations on ETD in combination with chip-based nanoelectrospray (nanoESI) as a platform for high throughput top-down proteomics are also presented.     

Utility of mass spectrometry for proteome ana lysis: part I. Conceptual and experimental approaches

This article is the first in a series of reviews intended as a tutorial providing the inexperienced, as well as the experienced, reader with an overview of principles of peptide and protein fragmentation in mass spectrometers for protein identification, surveying of the different types of instrument configurations and their combinations for protein identification. The first mass spectrometer was developed in 1899, but it took almost a century for the instrument to become a routine analytical method in proteomic research when fast atom bombardment ionization was developed, followed shortly by soft desorption/ionization methods, such as MALDI and electrospray ionization, to volatize biomolecules with masses of tens of kiloDaltons into the gas phase under vacuum pressure without destroying them. Thereafter, other soft ionization techniques that offered ambient conditions were also introduced, such as atmospheric pressure MALDI, direct analysis in real time, atmospheric-pressure solid analysis probe and hybrid ionization, sources of MALDI and electrospray ionization (e.g., two-step fused droplet electrospray ionization, laser desorption atmospheric-pressure chemical ionization, electrosonic spray ionization, desorption electrospray ionization, and electrospray-assisted laser desorption/ionization). The five basic types of mass analyzers currently used in proteomic research are the quadrupole, ion trap, orbitrap, Fourier transform ion cyclotron resonance and TOF instruments, which differ in how they determine the mass-to-charge ratios of the peptides. They have very different design and performance characteristics. These analyzers can be stand alone or, in some cases, put together in tandem or in conjunction with ion mobility mass spectrometry to take advantage of the strengths of each. Several singly or multiply charged fragment ion types, such as b, y, a, c, z, v, y and immonium ions are produced in the gas phase of the spectrometer. In the bottom-up sequencing approach for protein identification in a shotgun proteomic experiment, proteolytic digestion of proteins is accomplished by cleavage of the different bonds along the peptide backbone and/or side chain through a charge-directed transfer to the vicinity of the cleavage side. These various mass spectrometers and the types of ions produced have become important analytical tools for studying and analyzing proteins, peptides and amino acids.     

A tandem mass spectrometric study of bile acids: interpretation of fragmentation pathways and differentiation of steroid isomers

Bile acids are steroids with a pentanoic acid substituent at C-17. They are the terminal products of cholesterol excretion, and play critical physiological roles in human and animals. Bile acids are easy to detect but difficult to identify by using mass spectrometry due to their poly-ring structure and various hydroxylation patterns. In this study, fragmentation pathways of 18 free and conjugated bile acids were interpreted by using tandem mass spectrometry. The analyses were conducted on ion trap and triple quadrupole mass spectrometers. Upon collision-induced dissociation, the conjugated bile acids could cleave into glycine or taurine related fragments, together with the steroid skeleton. Fragmentations of free bile acids were further elucidated, especially by atmospheric pressure chemical ionization mass spectrometry in positive ion mode. Aside from universally observed neutral losses, eliminations occurred on bile acid carbon rings were proposed for the first time. Moreover, four isomeric 5β-cholanic acid hydroxyl derivatives (3α,6α-, 3α,7β-, 3α,7α-, and 3α,12α-) were differentiated using electrospray ionization in negative ion mode: 3α,7β-OH substituent inclined to eliminate H(2)O and CH(2)O(2) groups; 3α,6α-OH substituent preferred neutral loss of two H(2)O molecules; 3α,12α-OH substituent apt to lose the carboxyl in the form of CO(2) molecule; and 3α,7α-OH substituent exhibited no further fragmentation after dehydration. This study provided specific interpretation for mass spectra of bile acids. The results could contribute to bile acid analyses, especially in clinical assays and metabonomic studies.     

Evaluation of yellow rectangle traps coated with hot melt pressure sensitive adhesive and sticky gel against Rhagoletis indifferens (Diptera: Tephritidae)

Sticky yellow rectangle traps have been used for many years to capture Rhagoletis (Diptera: Tephritidae) fruit flies. Traditional sticky yellow traps are coated with a sticky gel (SG) that can leave residues on the hands of users. An alternative to SG on traps are hot melt pressure sensitive adhesives (HMPSAs), which are less messy. The main objective here was to evaluate two rectangle traps of two yellow colors, the Alpha Scents Yellow Card coated with HMPSA (Alpha Scents, West Linn, OR), and the Pherocon AM trap coated with SG (Pherocon; Trécé, Adair, OK), for capturing western cherry fruit fly, Rhagoletis indifferens Curran. Flies captured on both traps and held in the laboratory and field did not escape their surfaces. Flies caught on HMPSA were damaged when removed from traps without citrus solvent, whereas flies caught on SG could be removed intact without solvent. In field tests, Alpha Scents traps baited with an ammonium bicarbonate lure captured 1.4-2.2 times more R. indifferens than Pherocon traps baited with the same lure. Results of an experiment that eliminated differences in surface sticky material type, overall size, and surface sticky area between Alpha Scents and Pherocon traps suggested, although did not show conclusively, that more flies were caught on the Alpha Scents than Pherocon traps because of their different yellow color and/or lower fluorescence and not the HMPSA. Overall, the Alpha Scents trap is a viable alternative to the Pherocon trap for detecting R. indifferens.     

Recent advances in applications of liquid chromatography-tandem mass spectrometry to the analysis of reactive drug metabolites

Biotransformation of chemically stable compounds to reactive metabolites which can bind covalently to macromolecules, such as proteins and DNA, is considered as an undesirable feature of drug candidates. As part of an overall assessment of absorption, distribution, metabolism and excretion (ADME) properties, many pharmaceutical companies have put methods in place to screen drug candidates for their tendency to generate reactive metabolites and as well characterize the nature of the reactive metabolites through in vitro and in vivo studies. After identification of the problematic compounds, steps can be taken to minimize the potential of bioactivation through appropriate structural modifications. For these reasons, detection, structural characterization and quantification of reactive metabolites by mass spectrometry have become an important task in the drug discovery process. Triple quadrupole mass spectrometry is traditionally employed for the analysis of reactive metabolites. In the past 3 years, a number of new mass spectrometry methodologies have been developed to improve the sensitivity, selectivity and throughput of the analysis. This review focuses on the recent advances in the detection and characterization of reactive metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in drug discovery and development, especially through the use of linear ion trap (LTQ), hybrid triple quadrupole-linear ion trap (Q-trap) and the high resolution LTQ-Orbitrap instruments.     

Systematic comparison of a two-dimensional ion trap and a three-dimensional ion trap mass spectrometer in proteomics

The utility and advantages of the recently introduced two-dimensional quadrupole ion trap mass spectrometer in proteomics over the traditional three-dimensional ion trap mass spectrometer have not been systematically characterized. Here we rigorously compared the performance of these two platforms by using over 100,000 tandem mass spectra acquired with identical complex peptide mixtures and acquisition parameters. Specifically we compared four factors that are critical for a successful proteomic study: 1) the number of proteins identified, 2) sequence coverage or the number of peptides identified for every protein, 3) the data base matching SEQUEST X(corr) and S(p) score, and 4) the quality of the fragment ion series of peptides. We found a 4-6-fold increase in the number of peptides and proteins identified on the two-dimensional ion trap mass spectrometer as a direct result of improvement in all the other parameters examined. Interestingly more than 70% of the doubly and triply charged peptides, but not the singly charged peptides, showed better quality of fragmentation spectra on the two-dimensional ion trap. These results highlight specific advantages of the two-dimensional ion trap over the conventional three-dimensional ion traps for protein identification in proteomic experiments.     

Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations

Researchers have several options when designing proteomics experiments. Primary among these are choices of experimental method, instrumentation and spectral interpretation software. To evaluate these choices on a proteome scale, we compared triplicate measurements of the yeast proteome by liquid chromatography tandem mass spectrometry (LC-MS/MS) using linear ion trap (LTQ) and hybrid quadrupole time-of-flight (QqTOF; QSTAR) mass spectrometers. Acquired MS/MS spectra were interpreted with Mascot and SEQUEST algorithms with and without the requirement that all returned peptides be tryptic. Using a composite target decoy database strategy, we selected scoring criteria yielding 1% estimated false positive identifications at maximum sensitivity for all data sets, allowing reasonable comparisons between them. These comparisons indicate that Mascot and SEQUEST yield similar results for LTQ-acquired spectra but less so for QSTAR spectra. Furthermore, low reproducibility between replicate data acquisitions made on one or both instrument platforms can be exploited to increase sensitivity and confidence in large-scale protein identifications.     

Mass trapping of Prays nephelomima (Lepidoptera: Yponomeutidae) in citrus orchards: optimizing trap design and density

The moth Prays nephelomima (Meirick) (Lepidoptera: Yponomeutidae) is a significant pest of citrus (Citrus spp.), and the recent identification of the female sex pheromone has enabled new direct control tactics to be considered. Six trap designs were compared for suitability in mass trapping, and Pherocon III delta traps were chosen to further evaluate mass trapping. A mass trapping field trial was carried out at five lemon, Citrus limon L., orchards to determine the effect of trap density on catch and rind spot damage on fruit. One plot (0.33-1.0 ha) of each of the five trap density treatments (3, 10, 30, 100, and 300 traps/ha) were operated at each orchard over 12 wk. Catch per trap was reduced as trap density increased and a mean of 12,000 and 16,000 males per ha were killed at the trap densities of 100 and 300 traps per ha, respectively. Increased trap density reduced the percentage of flowers infested with P. nephelomima larvae and reduced the number of moths emerging from flowers. The incidence of rindspot damage on fruit decreased from 45 to 16% as the density of traps increased from 3 to 100 traps per ha. Incidence (percentage of fruit with rindspot) and severity (number of rindspots per fruit) was similar at 100 and 300 traps per ha, indicating that the optimal trap density for reducing rindspot damage is likely to be between 30 and 100 traps per ha. Prospects for converting mass trapping to a lure and kill system are discussed.     

Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes

In Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila.     

A simple solid phase mass tagging approach for quantitative proteomics

New mass-tagging reagents for quantitative proteomics measurements have been designed using solid phase peptide synthesis technology. The solid phase mass tags have been used to accurately measure the relative amounts of cysteine-containing peptides in model peptide mixtures as well as in mixtures of tryptic digests in the femtomol range. Measurements were made using both matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and online reversed-phase capillary liquid chromatography coupled through a nanoelectrospray interface to an ion trap mass spectrometer (capillary LC/ESI-MS). Results of mass-tagging experiments obtained from these two mass spectrometry techniques and their relative advantages and disadvantages for identification and quantitation of mass tagged peptides are compared. These reagents provide a simple, rapid and cost-effective alternative to currently available mass tagging technologies.     

Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications

The unique scanning capabilities of a hybrid linear ion trap (Q TRAP) mass spectrometer are described with an emphasis on proteomics applications. The combination of the very selective triple quadrupole based tandem mass spectrometry (MS/MS) scans with the very sensitive ion trap product ion scans allows rapid identification of peptides at low concentrations derived from post-translationally modified proteins on chromatographic time scales. The Q TRAP instrument also offers the opportunity to conduct a variety of ion processing steps prior to performing a mass scan. For example, the enhancement of the multiple-charge ion contents of the ion trap can be performed resulting in a survey mass spectrum dominated by double- and triple-charge peptides. This facilitates the identification of relevant biological species in both separated and unseparated peptide mixtures for further MS/MS experiments.     

Trade-off between high sensitivity and increased potential for false positive peptide sequence matches using a two-dimensional linear ion trap for tandem mass spectrometry-based proteomics

Two-dimensional linear ion trap mass spectrometers are rapidly becoming the new workhorse instruments for shotgun proteomic analysis of complex peptide mixtures. The objective of this study was to compare the potential for false positive peptide sequence matches between a two-dimensional ion trap instrument and a traditional, three-dimensional ion trap instrument. Through the comparative analysis of a complex protein sample, we found that in order to minimize false positive sequence matches, sequence match scoring criteria must be more stringent for data from the two-dimensional ion trap compared to the three-dimensional ion trap data. Given this increased potential for false positives, we also investigated two potential filtering strategies to reduce the false positive matches for data derived from the two-dimensional ion trap, including trypsin enzyme cleavage filtering, and the addition of peptide physicochemical information as a constraint, specifically peptide isoelectric point. The results described here provide a cautionary tale to researchers, demonstrating the need for careful analysis of MS/MS data from this new class of ion trap instruments, as well as the effectiveness of trypsin enzyme cleavage filtering and peptide pI information in maximizing high confidence protein identifications from this powerful proteomic instrumentation.     

Characterization of phosphorylation sites on histone H1 isoforms by tandem mass spectrometry

Histone H1 isoforms isolated from asynchronously grown HeLa cells were subjected to enzymatic digestion and analyzed by nano-flow reversed-phase high performance liquid chromatography (RP-HPLC) tandem mass spectrometry (MS/MS) on both quadrupole ion trap and linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometers. We have observed all five major isoforms of histone H1 (H1.1, H1.2, H1.3, H1.4, and H1.5) as well as a lesser studied H1, isoform H1.X. MS/MS experiments confirmed N-terminal acetylation on all isoforms plus a single internal acetylation site. Immobilized metal affinity chromatography in combination with tandem mass spectrometry was utilized to identify 19 phosphorylation sites on the five major H1 isoforms plus H1.X. Fourteen of these phosphorylation sites were located on peptides containing the cyclin dependent kinase (CDK) consensus motif (S/T)-P-X-Z (where X is any amino acid and Z is a basic amino acid). Five phosphorylation sites were identified in regions that did not fit the consensus CDK motif. One of these phosphorylation sites was found on the serine residue on the H1.4 peptide KARKSAGAAKR. The adjacent lysine residue to the phosphoserine was also shown to be methylated. This finding raises the question of whether the hypothesized "methyl/phos" switch could be extended to linker histones, and not exclusive to core histones.